(r)-3-((3s,4s)-3-fluoro-4-(4-hydroxyphenyl)piperidin-1-yl)-1-(4-methylbenzyl)pyrrolidin-2-one and its prodrugs for the treatment of psychiatric disorders

ABSTRACT

The disclosure generally relates to compounds of formula I, including their salts, as well as compositions and methods of using the compounds. The compounds are ligands for the NR2B NMDA receptor and may be useful for the treatment of various disorders of the central nervous system.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Continuation application claims the benefit of Ser. No. 15/971,049filed May 4, 2018, now pending, which is a Continuation applicationwhich claims priority to U.S. Ser. No. 15/846,914 filed Dec. 19, 2017,now abandoned, which is a Continuation application which claims priorityto U.S. Ser. No. 15/679,847 filed Aug. 17, 2017, now abandoned, which isa Continuation application which claims priority to U.S. Ser. No.15/490,558 filed Apr. 18, 2017, now abandoned, which is a Continuationapplication which claims priority to U.S. Ser. No. 15/357,102 filed Nov.21, 2016, now abandoned, which is a Continuation application whichclaims priority to U.S. Ser. No. 14/881,932 filed Oct. 13, 2015, nowabandoned, which is a Continuation application which claims the priorityof U.S. Ser. No. 14/591,372 filed Jan. 7, 2015, now U.S. Pat. No.9,187,506, which is a Non-Provisional application which claims thebenefit of Provisional application U.S. Ser. No. 61/925,405 filed Jan.9, 2014, now expired, hereby incorporated by reference in theirentireties.

BACKGROUND OF THE INVENTION

The disclosure generally relates to compounds of formula I, includingtheir salts, as well as compositions and methods of using the compounds.The compounds are ligands for the NR2B NMDA receptor and may be usefulfor the treatment of various disorders of the central nervous system.

N-Methyl-D-aspartate (NMDA) receptors are ion channels which are gatedby the binding of glutamate, an excitatory neurotransmitter in thecentral nervous system. They are thought to play a key role in thedevelopment of a number of neurological diseases, including depression,neuropathic pain, Alzheimer's disease, and Parkinson's disease.Functional NMDA receptors are tetrameric structures primarily composedof two NR1 and two NR2 subunits. The NR2 subunit is further subdividedinto four individual subtypes: NR2A, NR2B, NR2C, and NR2D, which aredifferentially distributed throughout the brain. Antagonists orallosteric modulators of NMDA receptors, in particular NR2Bsubunit-containing channels, have been investigated as therapeuticagents for the treatment of major depressive disorder (G. Sanacora,2008, Nature Rev. Drug Disc. 7: 426-437).

The NR2B receptor contains additional ligand binding sites in additionto that for glutamate. Non-selective NMDA antagonists such as Ketamineare pore blockers, interfering with the transport of Ca⁺⁺ through thechannel. Ketamine has demonstrated rapid and enduring antidepressantproperties in human clinical trials as an i.v. drug. Additionally,efficacy was maintained with repeated, intermittent infusions ofKetamine (Zarate et al., 2006, Arch. Gen. Psychiatry 63: 856-864). Thisclass of drugs, though, has limited therapeutic value because of its CNSside effects, including dissociative effects.

An allosteric, non-competitive binding site has also been identified inthe N-terminal domain of NR2B. Agents which bind selectively at thissite, such as Traxoprodil, exhibited a sustained antidepressant responseand improved side effect profile in human clinical trials as an i.v.drug (Preskorn et al., 2008, J. Clin. Psychopharmacol., 28: 631-637, andF. S. Menniti, et al., 1998, CNS Drug Reviews, 4, 4, 307-322). However,development of drugs from this class has been hindered by lowbioavailability, poor pharmacokinetics, and lack of selectivity againstother pharmacological targets including the hERG ion channel. Blockadeof the hERG ion channel can lead to cardiac arrythmias, including thepotentially fatal Torsades de pointe, thus selectivity against thischannel is critical. Thus, in the treatment of major depressivedisorder, there remains an unmet clinical need for the development ofeffective NR2B-selective negative allosteric modulators which have afavorable tolerability profile.

NR2B receptor antagonists have been disclosed in PCT publication WO2009/006437.

The invention provides technical advantages, for example, the compoundsare novel and are ligands for the NR2B receptor and may be useful forthe treatment of various disorders of the central nervous system.Additionally, the compounds provide advantages for pharmaceutical uses,for example, with regard to one or more of their mechanism of action,binding, inhibition efficacy, target selectivity, solubility, safetyprofiles, or bioavailability.

DESCRIPTION OF THE INVENTION

One aspect of the invention is a compound of formula I

where:Ar¹ is phenyl or indanyl and is substituted with 0-3 substituentsselected from cyano, halo, alkyl, haloalkyl, and haloalkoxy;Ar² is phenyl substituted with 1 OR substituent and also substitutedwith 0-3 substituents selected from cyano, halo, alkyl, haloalkyl, andhaloalkoxy;R is a prodrug moiety selected from the group consisting of alkylesters, amino acid esters, alkoxy esters, phosphonic acids, phosphonicalkyl esters, alkoxyphosphononate acid, alkoxyphosphonate alkyl esters,alkyl carabamates, amino acid carbamates, alkyl phosporamidates, arylphosphoramidates, and sulfamates;X is a bond or C₁-C₃ alkylene;n is 1 or 2; andring A is azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl,homopiperidinyl, or homopiperazinyl and is substituted with 0-4substituents selected from halo, alkyl, hydroxy, or alkoxy;or a pharmaceutically acceptable salt thereof.Another aspect of the invention is a compound of the formula

where R is a prodrug moiety selected from the group consisting of alkylesters, amino acid esters, alkoxy esters, phosphonic acids, phosphonicalkyl esters, alkoxyphosphononate acid, alkoxyphosphonate alkyl esters,alkyl carabamates, amino acid carbamates, alkyl phosporamidates, arylphosphoramidates, and sulfamates; or a pharmaceutically acceptable saltthereof.

Synthetic Methods

Compounds of Formula I may be made by methods known in the art includingthose described below and including variations within the skill of theart. Some reagents and intermediates are known in the art. Otherreagents and intermediates can be made by methods known in the art usingreadily available materials. The variables (e.g. numbered “R”substituents) used to describe the synthesis of the compounds areintended only to illustrate how to make the compounds and are not to beconfused with variables used in the claims or in other sections of thespecification. The following methods are for illustrative purposes andare not intended to limit the scope of the invention. The schemesencompass reasonable variations known in the art.

Scheme 1 shows an effective synthesis of example 1,(R)-3-((3S,4S)-3-fluoro-4-(4-hydroxyphenyl)piperidin-1-yl)-1-(4-methylbenzyl)pyrrolidin-2-one.Hydroxylactam 1 is available commercially in optically pure form. It canbe protected and N-alkylated to form lactam 4. Deprotection andactivation of the hydroxyl group with methanesulfonylchloride leads tothe lactam 5. Separately, compound 6 can be prepared by the Suzukicoupling reaction between commercial 4-benzyloxybromobenzene andcommercial tert-butyl4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6-dihydropyridine-1(2H)-carboxylate.Treatment of 6 with in-situ prepared borane followed by oxidationresults in formation of the trans racemic alcohol 7. The alcohol 7 canbe separated in to the individual enantiomers, and the phenol can beunmasked using hydrogenation under standard conditions to the preparethe substituted phenol 8. Fluorination with de-oxofluor reagent providesselectively the trans aryl fluoride 9, and deprotection of the Boc groupwith hydrochloric acid provides the piperidine as the hydrochloridesalt. Simple extraction under basic conditions provides the piperidine10 as the freebase. Careful reaction of the piperidine 10 with thelactam 5 under mildly basic conditions provides(R)-3-((3S,4S)-3-fluoro-4-(4-hydroxyphenyl)piperidin-1-yl)-1-(4-methylbenzyl)pyrrolidin-2-one,the title compound of example 1.

The compound of example 1 can be transformed into a variety of prodrugsusing methods known in the art. Thus, according to scheme 2, treatmentof the phenol with POCl₃, pyridine, and DMAP followed by aqueoushydrolysis provides example 2, the dihydrogen phosphate ester of example1.

Similarly, reaction of the compound of example 1 with a Boc-protectedamino acid using a variety of methods known in the art, but preferablyusing dicyclohexylcarbodiimide and 4-dimethylaminopyridine provides theester 11. Cleavage of the Boc group in acid, preferably HCl, providesthe esters which included the compounds of examples 3 and 4.

In a similar manner, Boc-protected aspartic acid tert-butyl ester (12)can be coupled through the unprotected sidechain to the compound ofexample 1 to provide the ester 13. Deprotection with HCl again providesthe compound of example 5.

Description of Specific Embodiments

Abbreviations used in the schemes generally follow conventions used inthe art. Chemical abbreviations used in the specification and examplesare defined as follows: “NaHMDS” for sodium bis(trimethylsilyl)amide;“DMF” for N,N-dimethylformamide; “MeOH” for methanol; “NBS” forN-bromosuccinimide; “Ar” for aryl; “TFA” for trifluoroacetic acid; “LAH”for lithium aluminum hydride; “BOC” for t-butoxycarbonyl, “DMSO” fordimethylsulfoxide; “h” for hours; “EtOAc” for ethyl acetate; “THF” fortetrahydrofuran; “EDTA” for ethylenediaminetetraacetic acid; “Et₂O” fordiethyl ether; “DMAP” for 4-dimethylaminopyridine; “DCE” for1,2-dichloroethane; “ACN” for acetonitrile; “DME” for1,2-dimethoxyethane; “HOBt” for 1-hydroxybenzotriazole hydrate; “DIEA”for diisopropylethylamine, “Nf” for CF₃(CF₂)₃SO₂—; and “TMOF” fortrimethylorthoformate.

Abbreviations as used herein, are defined as follows: “1×” for once,“2×” for twice, “3×” for thrice, “° C.” for degrees Celsius, “eq” forequivalent or equivalents, “g” for gram or grams, “mg” for milligram ormilligrams, “L” for liter or liters, “mL” for milliliter or milliliters,“μL” for microliter or microliters, “N” for normal, “M” for molar,“mmol” for millimole or millimoles, “min” for minute or minutes, “h” forhour or hours, “rt” for room temperature, “RT” for retention time, “atm”for atmosphere, “psi” for pounds per square inch, “conc.” forconcentrate, “sat” or “satd.” for saturated, “MW” for molecular weight,“mp” for melting point, “ee” for enantiomeric excess, “MS” or “MassSpec” for mass spectrometry, “ESI” for electrospray ionization massspectroscopy, “HR” for high resolution, “HRMS” for high resolution massspectrometry, “LCMS” for liquid chromatography mass spectrometry, “HPLC”for high pressure liquid chromatography, “RP HPLC” for reverse phaseHPLC, “DCM” for dichloromethane, “TLC” or “tlc” for thin layerchromatography, “SFC” for supercritical fluid chromatography, “NMR” fornuclear magnetic resonance spectroscopy, “¹H” for proton, “□” for delta,“s” for singlet, “d” for doublet, “t” for triplet, “q” for quartet, “m”for multiplet, “br” for broad, “Hz” for hertz, and “R”, “S”, “E”, and“Z” are stereochemical designations familiar to one skilled in the art.

LC/MS data were acquired using the following conditions:Conditions A: Ascentis C18 50×2.1 mm, 2.7 μm column using a 1 mL/minflowrate gradient of 0-100% B over 1.7 minutes followed by 1.3 minutesat 100% B. Solvent A: 10 mM NH4COOH in water:acetonitrile (98:2);solvent B=10 mM NH4COOH in water:acetonitrile (2:98).Conditions B: Phenomenex C18 2.0×50 mm, 5 μm column using a 0.8 mL/minflowrate gradient of 0-100% B over 4 minutes. Solvent A=10% MeOH/90%water/0.1% TFA, Solvent B=90% MeOH/10% water/0.1% TFA.

Synthesis of Intermediates Intermediate A. tert-Butyl4-(4-(benzyloxy)phenyl)-5,6-dihydropyridine-1(2H)-carboxylate

A solution of commercial 1-(benzyloxy)-4-bromobenzene (104 g, 395 mmol)and commercial tert-butyl4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6-dihydropyridine-1(2H)-carboxylate(147 g, 474 mmol) in 1100 mL of acetonitrile was purged with nitrogenfor 2 min. Water (1100 mL) was added, followed by sodium carbonate (126g, 1186 mmol) and tetrakis(triphenylphosphine)palladium (27.4 g, 23.7mmol). The reaction mixture was purged with nitrogen for 5 min, and thenheated to 90° C. and stirred for 16 h. The reaction mixture was thenallowed to cool to rt and diluted with 1 L of ethyl acetate. The layerswere separated, and the aqueous layer was extracted with two additional250 mL portions of ethyl acetate. The organic layers were combined,washed with 200 mL of brine, dried over sodium sulfate, and evaporatedin vacuo to provide an off-white solid. The product was purified bysilica gel chromatography eluting with 6% ethyl acetate in petroleumether to provide 129 g (88%) of the desired product. LC/MS RT(conditions A)=2.732 min, (M−H)+=364.0. ¹H NMR (300 MHz, chloroform-d) δ7.49-7.30 (m, 5H), 7.27 (d, J=10.7 Hz, 2H), 6.99-6.87 (m, 2H), 6.03-5.87(m, 1H), 5.07 (s, 2H), 4.05 (d, J=2.6 Hz, 2H), 3.62 (t, J=5.7 Hz, 2H),2.49 (br. s., 2H), 1.49 (s, 9H).

Intermediate B. (+/−)-rel-(3S,4S)-tert-Butyl4-(4-(benzyloxy)phenyl)-3-hydroxypiperidine-1-carboxylate

Sodium borohydride (15.5 g, 410 mmol) was dissolved in THF, and thesolution was chilled to 0° C. Boron trifluoride etherate (52.3 mL, 424mmol) was added to the solution and the mixture was allowed to warm tort and stirred for 30 min. Then a solution of tert-butyl4-(4-(benzyloxy)phenyl)-5,6-dihydropyridine-1(2H)-carboxylate (50 g, 137mmol, intermediate A) in 500 mL of THF was added and the reactionmixture was stirred for 2 h at rt. A 100 mL portion of water was thenadded slowly to the mixture (Caution: effervescence is observed). Themixture was diluted with 100 mL of ethanol, and sodium hydroxide (228mL, 10% solution in water, 0.684 mol) and hydrogen peroxide (20.5 mL,0.684 mol) were added. The reaction mixture was heated to refluxtemperature and stirred for 16 h. The mixture was cooled to 10° C. anddiluted with 1 L of DCM. Then the pH was adjusted to 7 with 1.5 L of 1.5N HCl. The layers were then separated, and the aqueous layer wasextracted with an addition two 500 mL portions of DCM. The organiclayers were combined, washed with 2×1 L of water and 200 mL of brine,dried over sodium sulfate, and evaporated in vacuo to provide anoff-white solid. The solid was triturated with 500 mL of pet ether, andisolated by filtration to yield 46.5 grams of product (88%, 99.0% purityby HPLC). LC/MS RT (conditions A)=2.372 min, (M+H)⁺=382.0. ¹H NMR (400MHz, DMSO-d₆) δ 7.47-7.42 (m, 2H), 7.42-7.36 (m, 2H), 7.36-7.28 (m, 1H),7.14 (d, J=9.0 Hz, 2H), 6.92 (d, J=9.0 Hz, 2H), 5.07 (s, 2H), 4.74 (d,J=5.5 Hz, 1H), 4.10 (br. s., 1H), 3.94 (br. s., 1H), 3.46-3.35 (m, 1H),2.47-2.31 (m, 2H), 1.70-1.61 (m, 1H), 1.55-1.45 (m, 2H), 1.42 (s, 9H).

Intermediate C. (3S,4S)-tert-Butyl4-(4-(benzyloxy)phenyl)-3-hydroxypiperidine-1-carboxylate

Racemic rel-(3S,4S)-tert-butyl4-(4-(benzyloxy)phenyl)-3-hydroxypiperidine-1-carboxylate (112 g,intermediate B) was separated into the individual enantiomers usingpreparative supercritical fluid chromatography under the followingconditions: A Thar SFC-250 instrument was utilized with aLux-Cellulose-2 (250×21 mm), 5 μm column eluting with 60% CO₂ and 40% ofa solution of 0.3% diethylamine in methanol at a flow rate of 100.0g/min. Sample was injected at 74 mg/mL. Analytical SFC was carried outon Lux-Cellulose-2 (250×4.6 mm), 5 μm column eluting with 55% CO₂ and45% of a solution of 0.3% diethylamine in methanol at a flow rate of 3.0g/min. The recovery was 50.0 g of peak 1 with a retention time of 2.49minutes, which corresponds to the desired (3S,4S)-tert-butyl4-(4-(benzyloxy)phenyl)-3-hydroxypiperidine-1-carboxylate. Analyticaldata matched those from the racemate.

Intermediate D. (3S,4S)-tert-Butyl3-hydroxy-4-(4-hydroxyphenyl)piperidine-1-carboxylate

A solution of (3S,4S)-tert-butyl4-(4-(benzyloxy)phenyl)-3-hydroxypiperidine-1-carboxylate (26 g, 67.8mmol, intermediate C) in 260 mL of methanol was treated with 1.6 gramsof 10% palladium on carbon (13.6 mmol) in a pressure bottle. Hydrogen at50 psi was introduced, and the reaction mixture was stirred for 16 h.The mixture was filtered through celite and concentrated to a crudeproduct (18.9 g, 64.4 mmol) which was sufficiently pure to carry forwardwithout further purification. LC/MS RT (conditions B)=2.970 min, (M+Hwith loss of t-butyl)⁺=238.0. ¹H NMR (400 MHz, DMSO-d₆) δ 9.10 (br. s.,1H), 7.01 (d, J=8.5 Hz, 2H), 6.65 (s, 2H), 4.70 (d, J=5.0 Hz, 1H), 4.09(br. s., 1H), 3.93 (br. s., 1H), 3.17 (s, 2H), 2.79-2.63 (m, 1H), 2.34(br. s., 1H), 1.68-1.57 (m, 1H), 1.44 (br. s., 1H), 1.42 (s, 9H).

Intermediate E. (3S,4S)-tert-Butyl3-fluoro-4-(4-hydroxyphenyl)piperidine-1-carboxylate

A solution of (3S,4S)-tert-butyl3-hydroxy-4-(4-hydroxyphenyl)piperidine-1-carboxylate (15.5 g, 61.4mmol, intermediate D) in 270 mL of acetonitrile was chilled to 0° C. Tothe stirred solution was added bis(2-methoxyethyl)aminosulfurtrifluoride 50% solution in toluene (Deoxo-fluor, 58.4 mL, 159 mmol)dropwise via addition funnel over 65 min. After the addition, thereaction mixture was stirred for 30 min at 0° C. and then allowed tocome to rt and stirred for an additional 2 h. A saturated ammoniumchloride solution (150 mL) was then added, and the mixture was extractedwith two 150 mL portions of DCM. The organic layers were combined, driedover sodium sulfate, and concentrated to afford the crude product. Theproduct was purified by silica gel chromatography (1.5 kg of silica)eluting with a gradient of 0-15% acetone in hexanes to afford 11.9 g(75%) of the desired (3S,4S)-tert-butyl3-fluoro-4-(4-hydroxyphenyl)piperidine-1-carboxylate. LC/MS RT(conditions B)=3.295 min, (M+H with loss of t-butyl and elimination offluorine)⁺=220.0. ¹H NMR (400 MHz, chloroform-d) δ 7.15 (d, J=8.6 Hz,2H), 6.83 (dt, J=8.6, 2.0 Hz, 2H), 4.59-4.48 (m, 1H), 4.47-4.37 (m, 1H),4.23-4.12 (m, 1H), 2.88-2.68 (m, 3H), 1.96-1.84 (m, 1H), 1.80-1.66 (m,1H), 1.51 (s, 9H).

Intermediate F. 4-((3S,4S)-3-Fluoropiperidin-4-yl)phenol

A solution of (3S,4S)-tert-butyl3-fluoro-4-(4-hydroxyphenyl)piperidine-1-carboxylate (12.0 g, 40.6 mmol,intermediate E) in anhydrous dioxane (80 mL) was treated with HCl (4 Min 1,4-dioxane, 40.6 mL, 162 mmol). The reaction mixture was allowed tostir at rt for 6 h and then evaporated in vacuo to provide the HCl saltof the desired product. Without further isolation, the HCl salt wassuspended in CHCl₃ and 80 mL of a satd. NaHCO₃ solution was added. Theorganic layer was separated, and the aqueous layer was extracted withCHCl₃ (2×100 mL). The organic layers were combined, dried over Na₂SO₄and concentrated to give the title compound (7.1 g, 36.4 mmol, 90%).LC/MS RT (conditions B)=1.008 min, LC/MS (M+H)⁺=196.2.

Intermediate G. (S)-3-((tert-Butyldimethylsilyl)oxy)pyrrolidin-2-one

A stirred solution of commercial (S)-3-hydroxypyrrolidin-2-one (5 g,49.5 mmol) in DCM (198 ml) was treated with DMAP (0.199 g, 1.632 mmol),imidazole (6.73 g, 99 mmol), and TBDMS-Cl (8.94 g, 59.3 mmol). Thereaction mixture was stirred at rt for 16 h, and then was washed with asatd. NaHCO₃ solution. The organic layer was concentrated and the crudereaction product was purified by silica gel chromatography eluting with50% ethyl acetate in petroleum ether. The desired product was isolatedas a white solid (8.1 g, 76%). LC/MS (M+H)⁺=216.2. ¹H NMR (400 MHz,chloroform-d) δ 6.40 (br. s., 1H), 4.26 (t, J=7.8 Hz, 1H), 3.42-3.34 (m,1H), 3.29-3.21 (m, 1H), 2.36 (dtd, J=12.7, 7.3, 3.3 Hz, 1H), 2.07-1.96(m, 1H), 0.91 (s, 9H), 0.15 (d, J=7.0 Hz, 6H).

Intermediate H.(S)-3-((tert-Butyldimethylsilyl)oxy)-1-(4-methylbenzyl)pyrrolidin-2-one

(S)-3-((tert-butyldimethylsilyl)oxy)pyrrolidin-2-one (5 g, 23.22 mmol,intermediate G) was dissolved in anhydrous THF (46.4 ml) and thereaction mixture was cooled to 0° C. under a nitrogen atmosphere. Sodiumhydride (1.393 g, 34.8 mmol) was then added in one portion and thereaction mixture was allowed to stir for 5 min before the dropwiseaddition of 1-(bromomethyl)-4-methylbenzene (5.37 g, 29.0 mmol) inanhydrous THF (46.4 ml). The reaction was allowed to stir at 0° C. for 5min, then the cooling bath was removed and mixture was allowed to warmto rt overnight. The reaction was cautiously quenched with water (100mL) and then extracted with ethyl acetate (3×100 mL). The combinedorganic layers were then washed with brine (200 mL) and dried (MgSO₄).Evaporation of the solvent in vacuo gave the crude product (9.6 g, oil)which was then purified by silica gel chromatography (330 g of silica)eluting with a gradient of 0% to 20% ethyl acetate in hexanes to provide6.53 g (88%) of the desired product. LC/MS (Conditions B), RT=4.320 min,(M+H)⁺=320.3. ¹H NMR (400 MHz, chloroform-d) δ 7.15 (s, 4H), 4.42 (s,2H), 4.37 (t, J=7.6 Hz, 1H), 3.32-3.18 (m, 1H), 3.10 (dt, J=9.7, 7.5 Hz,1H), 2.36 (s, 3H), 2.29 (dtd, J=12.6, 7.6, 3.1 Hz, 1H), 1.97-1.84 (m,1H), 0.95 (s, 9H), 0.20 (d, J=10.3 Hz, 6H).

Intermediate I. (S)-3-Hydroxy-1-(4-methylbenzyl)pyrrolidin-2-one

HCl (4 M in 1,4-dioxane, 25.5 ml, 102 mmol) was added in one portion toa solution of(S)-3-((tert-butyldimethylsilyl)oxy)-1-(4-methylbenzyl)pyrrolidin-2-one(6.53 g, 20.44 mmol, intermediate H) in anhydrous DCM (20.4 mL) at rt. Aslight exotherm was noted. The reaction mixture was allowed to stir atrt for 2 h and then evaporated in vacuo. The residue was taken up in DCM(100 mL) and washed with a satd. sodium bicarbonate solution (100 mL)and brine (50 mL), and then the solution was dried over MgSO₄ andconcentrated to a residue. The crude product was purified by silica gelchromatography (120 g of silica) eluting with a gradient of 40% to 100%ethyl acetate in hexanes to provide 3.73 g (89%) of the desired product.LC/MS (Conditions B), RT=2.338 min, (M+H)⁺=206.2. ¹H NMR (400 MHz,chloroform-d) δ 7.26-7.02 (m, 4H), 4.43 (d, J=3.5 Hz, 2H), 4.41-4.37 (m,1H), 3.66 (d, J=2.6 Hz, 1H), 3.34-3.05 (m, 2H), 2.41 (dddd, J=12.8, 8.4,6.6, 2.2 Hz, 1H), 2.34 (s, 3H), 1.93 (dq, J=12.8, 8.8 Hz, 1H).

Intermediate J. (S)-1-(4-methylbenzyl)-2-oxopyrrolidin-3-ylmethanesulfonate

Triethylamine (0.509 ml, 3.65 mmol) was added to a cooled solution of(S)-3-hydroxy-1-(4-methylbenzyl)pyrrolidin-2-one (0.5 g, 2.436 mmol,intermediate I) in anhydrous DCM (12.18 ml) at 0° C. under a nitrogenatmosphere. Methanesulfonyl chloride (0.198 ml, 2.56 mmol) was thenadded dropwise and the reaction was allowed to stir at 0° C. for 15 minbefore quenching with a satd. sodium bicarbonate solution (10 mL). Themixture was allowed to warm to rt and the aqueous layer was separatedand extracted with DCM (2×). The combined organic layers were dried overMgSO₄ and evaporated in vacuo to give a white solid (0.73 g) which wasthen purified by silica gel chromatography (40 g of silica) eluting witha gradient of 0% to 50% ethyl acetate in hexanes to provide 0.63 g (91%)of the desired product as a white solid.

Intermediate K.(S)-3-(tert-Butyldimethylsilyloxy)-1-(4-(difluoromethyl)benzyl)pyrrolidin-2-one

A 60% dispersion of sodium hydride in mineral oil (232 mg, 5.31 mmol)was added to a stirred solution of(S)-3-((tert-butyldimethylsilyl)oxy)pyrrolidin-2-one (762 mg, 3.54 mmol,intermediate G) in THF (7 mL) at 0° C. After 15 min, a solution of1-(bromomethyl)-4-(difluoromethyl)benzene (980 mg, 4.43 mmol) in THF (7mL) was added to the reaction mixture. The resulting mixture was stirredat room temperature for 6 h. The reaction was carefully quenched withseveral grams of ice pellets. The resulting mixture was extracted withEtOAc. The combined organic layers were washed with water, dried oversodium sulfate, filtered and concentrated in vacuo. The crude reactionmixture was purified using silica gel column chromatography (0-30%EtOAc/hexanes) to afford the desired product (440 mg, 35% yield) as awhite solid: LCMS (M+H)⁺ 356.3; ¹H NMR (500 MHz, chloroform-d) δ 7.49(d, J=8.1 Hz, 2H), 7.35 (d, J=7.9 Hz, 2H), 6.65 (br. t, J=1.0 Hz, 1H),4.56-4.44 (m, 2H), 4.38 (t, J=7.5 Hz, 1H), 3.27 (ddd, J=9.7, 8.7, 3.4Hz, 1H), 3.13 (dt, J=9.7, 7.4 Hz, 1H), 2.36-2.27 (m, 1H), 1.98-1.90 (m,1H), 0.96 (br. s., 9H), 0.22-0.20 (m, 3H), 0.20-0.18 (m, 3H).

Intermediate L.(S)-1-(4-(Difluoromethyl)benzyl)-3-hydroxypyrrolidin-2-one

A solution of 4 M HCl in dioxane (0.62 mL, 2.5 mmol) was added to astirred solution of(S)-3-((tert-butyldimethylsilyl)oxy)-1-(4-(difluoromethyl)benzyl)pyrrolidin-2-one(440 mg, 1.24 mmol, intermediate K) in dichloromethane (1.24 mL) at rt.The reaction mixture was stirred for 2 h. The reaction mixture wasconcentrated in vacuo to afford the desired product (368 mg,quantitative yield): LC-MS (M+H)⁺ 242.1.

Intermediate M. (S)-1-(4-(Difluoromethyl)benzyl)-2-oxopyrrolidin-3-ylMethanesulfonate

Triethylamine (0.319 mL, 2.29 mmol) and methansulfonyl chloride (0.131mL, 1.68 mmol) was added to a stirred solution of(S)-1-(4-(difluoromethyl)benzyl)-3-hydroxypyrrolidin-2-one (368 mg, 1.53mmol, intermediate L) in dichloromethane (7.63 mL) at 0° C. The reactionmixture was stirred at 0° C. for 1 h. The resulting mixture was dilutedwith water and the aqueous mixture was extracted with dichloromethane.The combined organic layers were washed with 10% sodium bicarbonatesolution, dried over sodium sulfate, filtered, and concentrated invacuo. The crude material was purified using silica gel columnchromatography (0-100% EtOAc). The pure fractions were combined andconcentrated in vacuo to afford the desired product (322 mg, 66% yield)as a white solid: LC-MS (M+H)⁺ 320.1; ¹H NMR (500 MHz, chloroform-d) δ7.53 (d, J=7.9 Hz, 2H), 7.38-7.33 (m, 2H), 6.67 (br. t, J=1.0 Hz, 1H),5.27 (dd, J=8.2, 7.5 Hz, 1H), 4.60-4.49 (m, 2H), 3.41-3.35 (m, 1H), 3.33(s, 3H), 3.27 (dt, J=9.9, 7.3 Hz, 1H), 2.64-2.55 (m, 1H), 2.27 (ddt,J=13.9, 8.9, 7.1 Hz, 1H).

Intermediate N. tert-Butyl4-hydroxy-4-(4-methoxyphenyl)piperidine-1-carboxylate

A mixture of commercial tert-butyl 4-oxopiperidine-1-carboxylate (2.0 g,10.0 mmol) and diethyl ether (30 ml) was cooled to 0° C. To this mixturewas added dropwise a solution of (4-methoxyphenyl)magnesium bromide (0.5M in diethyl ether, 30 ml, 15 mmol). After complete addition, thereaction mixture was allowed to warm to rt and stirred for 2 h. It wasthen slowly quenched with 150 ml of ice cold water and then theresulting mixture was extracted with 3×150 ml of DCM. The organic layerswere combined, dried, filtered, and concentrated under vacuum. The crudeproduct was purified by silica gel column chromatography (30:70 ethylacetate:hexane) to provide the desired product (3.0 g, 100% yield):LC-MS (ES-API): m/z 305.5 (M−H)⁺; ¹H NMR (400 MHz, DMSO-d₆) δ 7.37 (q,J=1.0 Hz, 2H), 6.86 (q, J=1.0 Hz, 2H), 4.94 (s, 1H), 3.82 (d, J=11.5 Hz,2H), 3.73 (s, 3H), 3.13 (br. s, 2H), 1.75 (td, J=12.9, 4.8 Hz, 2H), 1.56(d, J=12.3 Hz, 2H), 1.41 (s, 9H).

Intermediate O. 4-(4-Methoxyphenyl)-1,2,3,6-tetrahydropyridineHydrochloride

A mixture of tert-butyl4-hydroxy-4-(4-methoxyphenyl)piperidine-1-carboxylate (700 mg, 2.27mmol, intermediate N) and HCl in dioxane (4.0 ml, 16 mmol) was stirredat rt for 3 h. The crude mass was concentrated under vacuum and thesolid residue was washed with 3×10 ml of DCM to remove non-polarimpurities. The desired salt was collected as a fine solid (480 mg,93%). LCMS (ES-API) m/z 190.2 (M+H)⁺; ¹H NMR (400 MHz, DMSO-d₆) δ 7.37(d, J=9.0 Hz, 2H), 6.98 (d, J=9.0 Hz, 2H), 6.08-5.98 (m, 1H), 5.11 (s,1H), 3.97 (br. s., 1H), 3.52 (s, 1H), 3.32 (s, 3H), 2.47-2.37 (m, 1H).

Intermediate P. 4-(4-Methoxyphenyl)piperidine Hydrochloride

To a stirred solution of 4-(4-methoxyphenyl)-1,2,3,6-tetrahydropyridine,HCl (3.00 g, 13.3 mmol, intermediate O) in methanol (20 mL) was added10% palladium on carbon (1.4 g) and the reaction mixture was stirred at20 psi of hydrogen for 12 h. The reaction mixture was filtered through apad of celite, which was washed with ethyl acetate, and the combinedorganic fractions were concentrated to obtain a white solid (2.0 g, 70%yield): LCMS (ES-API), m/z 192.1 (M+H)⁺; ¹H NMR (300 MHz, DMSO-d₆) δ9.13-8.36 (m, 2H), 7.14 (d, J=8.7 Hz, 2H), 6.90 (d, J=8.7 Hz, 2H), 3.73(s, 3H), 3.07-2.87 (m, 4H), 2.87-2.65 (m, 4H).

Intermediate Q. 2,4-Dibromo-N-(4-fluorobenzyl)butanamide

TEA (8.91 mL, 63.9 mmol) and 2,4-dibromobutanoyl chloride (5.07 mL, 38.4mmol) were sequentionall added to solution of commercial(4-fluorophenyl)methanamine (4.0 g, 32.0 mmol) in diethyl ether (15 mL)at 0° C. The reaction mixture was allowed to warm to rt and stir for anadditional 24 h. The reaction mixture was filtered. The solids werewashed with diethyl ether. The filtrate was concentrated in vacuo toafford a crude mixture containing2,4-dibromo-N-(4-fluorobenzyl)butanamide (8.0 g, 71% yield): LCMS(ES-API), m/z 354, 356 (M+H)⁺.

Intermediate R. 3-Bromo-1-(4-fluorobenzyl)pyrrolidin-2-one

A 60% dispersion of NaH in mineral oil (1.70 g, 42.5 mmol) was added toa stirred solution of 2,4-dibromo-N-(4-fluorobenzyl)butanamide (10.0 g,28.3 mmol, intermediate Q) in THF (25 mL) at 0° C. The reaction mixturewas allowed to warm to rt and stir for and additional 2 h. The reactionmixture was carefully quenched with ice and diluted with water. Theresulting mixture was extracted with EtOAc. The combined organic layerswere washed with water and then brine solution. The organic layer wasover sodium sulfate, filtered, and concentrated in vacuo. The crudeproduct was purified using silica gel column chromatography (10%EtOAc/hexanes) to afford the desired product (5.90 g, 64% yield): LCMS(ES-API), m/z 272.4, 274.3 (M+H)⁺; ¹H NMR (400 MHz, DMSO-d₆) δ ppm2.12-2.27 (m, 1H) 2.56-2.68 (m, 1H) 3.27 (dd, J=7.78, 3.26 Hz, 2H)4.29-4.38 (m, 1H) 4.40-4.57 (m, 1H) 4.73 (dd, J=7.03, 3.01 Hz, 1H)7.04-7.35 (m, 4H).

Intermediate S.1-(4-Fluorobenzyl)-3-(4-(4-methoxyphenyl)piperidin-1-yl)pyrrolidin-2-one

TEA (0.768 mL, 5.51 mmol) was added to a stirred solution of3-bromo-1-(4-fluorobenzyl)pyrrolidin-2-one (0.3 g, 1.10 mmol,intermediate R) and 4-(4-methoxyphenyl)piperidine hydrochloride (0.276g, 1.213 mmol, intermediate P) in acetonitrile (10 mL). The reactionmixture was sealed and heated in a chemistry microwave at 100° C. for 1h. The reaction mixture was cooled to rt and concentrated in vacuo. Theresidue was diluted with EtOAc. The organic mixture was washed withwater and brine solution. The organic layer was dried over sodiumsulfate, filtered and concentrated in vacuo to afford a crude mixturecontaining1-(4-fluorobenzyl)-3-(4-(4-methoxyphenyl)piperidin-1-yl)pyrrolidin-2-one(0.35 g, 83% yield): LCMS (ES-API), m/z 383.2 (M+H)⁺.

Example 1.(R)-3-((3S,4S)-3-fluoro-4-(4-hydroxyphenyl)piperidin-1-yl)-1-(4-methylbenzyl)pyrrolidin-2-one

A solution of 4-((3S,4S)-3-fluoropiperidin-4-yl)phenol (7.10 g, 36.4mmol, intermediate F) and DIEA (16 mL, 92 mmol) in 100 mL ofacetonitrile was heated to 80° C. This solution was treated dropwisewith a solution of (S)-1-(4-methylbenzyl)-2-oxopyrrolidin-3-ylmethanesulfonate (10.5 g, 37.0 mmol, intermediate J) in acetonitrile (80mL) over a period of 4 hours. After the addition was completed, thereaction mixture was stirred at 80° C. for 16 h. The reaction mixturewas then allowed to cool to rt, and the volume was reduced by rotaryevaporation to 80 mL. A satd. NH₄Cl solution (100 mL) was then added,and the layers were separated. The aqueous layer was extracted with DCM(2×100 mL) and the organic layers were combined, dried over Na₂SO₄ andconcentrated in vacuo to give a crude product. The crude product waspurified by silica gel chromatography (750 g of silica gel) eluting witha gradient of 0% to 20% of solvent B in solvent A, where Solvent B=20%methanol/DCM and solvent A=DCM. Fractions containing the product werecombined. Evaporation of the solvents gave 9.3 grams of the desiredproduct with 97% purity by LC/MS analysis (conditions B). The productthus obtained (8.5 g) was slurried in acetone:hexane (1:5, 200 mL) andthe solid product was isolated by filtration and air dried. Careful SFCanalysis showed the presence of a 2.1% impurity in the product. Using aCell4 0.46×25 cm 5 μm column and eluting with 45% methanol in CO₂ at 3mL/min, the desired product eluted at 3.800 minutes and the undesiredimpurity eluted at 4.848 minutes. The product was then further purifiedby SFC Chromatography using a Cell4 3×25 cm 5 μm column at 150 mL/mininjecting 1.5 mL of a 80 mg/mL solution. Concentration of the activefractions provided 7.82 grams (20.4 mmol, 56%) of >99.7% pure example 1as a white powder. LC/MS (Conditions B), RT=2.512 min, (M+H)⁺=383.3. ¹⁹FNMR δ −182.83. ¹H NMR (400 MHz, chloroform-d) δ 7.20-7.08 (m, 6H),6.98-6.78 (m, 2H), 5.68 (s, 1H), 4.77-4.54 (m, 1H), 4.53-4.34 (m, 2H),3.68 (t, J=8.8 Hz, 1H), 3.41-3.29 (m, 1H), 3.28-3.09 (m, 2H), 2.82 (d,J=10.8 Hz, 1H), 2.74-2.54 (m, 2H), 2.47 (td, J=9.9, 3.6 Hz, 1H), 2.34(s, 3H), 2.19-1.94 (m, 2H), 1.92-1.80 (m, 2H). ¹³C NMR (101 MHz,chloroform-d) δ 172.4, 154.9, 137.5, 133.3, 133.0, 129.5, 128.7, 128.3,115.5, 92.6, 90.8, 65.0, 54.4, 54.2, 48.7, 48.0, 47.8, 46.8, 43.7, 31.7,31.6, 21.1, 19.6.

Example 2.4-((3S,4S)-3-Fluoro-1-((R)-1-(4-methylbenzyl)-2-oxopyrrolidin-3-yl)piperidin-4-yl)phenylDihydrogen Phosphate

To a suspension of(R)-3-((3S,4S)-3-fluoro-4-(4-hydroxyphenyl)piperidin-1-yl)-1-(4-methylbenzyl)pyrrolidin-2-one(100 mg, 0.261 mmol, example 1) in 10 mL of dichloromethane was addedpyridine (0.106 mL, 1.31 mmol) and DMAP (160 mg, 1.31 mmol). Thereaction mixture was chilled to −20° C. To the chilled solution wasadded POCl₃ (0.122 mL, 1.31 mmol) dropwise, and then the reactionmixture was allowed to warm to rt and stirred for 1 h. Water (10 mL) wasadded and the mixture was stirred for 1.5 h. The layers were thenseparated and the organic layer was dried over Na₂SO₄ and evaporated todryness. The crude product was purified by HPLC on a Symmetry C8 (300×17mm) 7 mM column eluting with a gradient of 20% B to 50% B over 7 minutesat 15 mL/min where solvent A=10 mM ammonium acetate in water pH 4.5 andsolvent B=acetonitrile. The product RT=2.2 min. The desired product (5.8mg, 4.7%) was isolated from the appropriate fractions by lyophilizationas a white solid. LCMS (Conditions A) RT=1.720 min, (M+H)⁺=463.2. ¹H NMR(400 MHz, methanol-d4) δ 7.29-7.16 (m, 8H), 4.74 (br. s., 1H), 4.61-4.34(m, 2H), 4.01 (t, J=8.3 Hz, 1H), 3.82-3.62 (m, 1H), 3.35 (m, 2H), 3.05(br. s., 2H), 2.79 (br. s., 2H), 2.34 (s, 4H), 2.18 (br. s., 1H),2.02-1.87 (m, 1H), 1.83 (br. s., 1H). ¹⁹F NMR (376 MHz, methanol-d4) δ−185.143. ³¹P NMR (162 MHz, methanol-d4) δ −4.260.

Example 3.(S)-4-((3S,4S)-3-Fluoro-1-((R)-1-(4-methylbenzyl)-2-oxopyrrolidin-3-yl)piperidin-4-yl)phenyl2-amino-3-methylbutanoate Hydrochloride

Step 3A.(S)-4-((3S,4S)-3-Fluoro-1-((R)-1-(4-methylbenzyl)-2-oxopyrrolidin-3-yl)piperidin-4-yl)phenyl2-((tert-butoxycarbonyl)amino)-3-methylbutanoate

To a solution of(R)-3-((3S,4S)-3-fluoro-4-(4-hydroxyphenyl)piperidin-1-yl)-1-(4-methylbenzyl)pyrrolidin-2-one(0.02 g, 0.052 mmol, example 1) in DCM (3 mL) was added(S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanoic acid (0.059 g, 0.272mmol) followed by DCC (0.032 g, 0.157 mmol) and DMAP (6.39 mg, 0.052mmol). The reaction mixture was stirred at room temperature for 18 h.Water (10 mL) was then added, and the layers were separated. The aqueouslayer was extracted with DCM (3×10 mL) and the organic layers werecombined, dried over Na₂SO₄, and concentrated to a crude product. Thecrude product was purified by preparative TLC eluting with 35% ethylacetate in petroleum ether to provide the purified product(S)-4-((3S,4S)-3-fluoro-1-((R)-1-(4-methylbenzyl)-2-oxopyrrolidin-3-yl)piperidin-4-yl)phenyl2-((tert-butoxycarbonyl)amino)-3-methylbutanoate (27 mg, 79%). LC/MS(Conditions A) RT=2.523 min, (M+H)⁺=582.2. ¹H NMR (400 MHz, methanol-d₄)δ 7.36 (d, J=8.5 Hz, 2H), 7.18 (s, 4H), 7.08 (d, J=8.5 Hz, 2H),4.80-4.59 (m, J=10.0, 10.0, 5.0 Hz, 1H), 4.51 (d, J=15.0 Hz, 1H), 4.39(d, J=15.0 Hz, 1H), 4.23 (dd, J=8.3, 6.3 Hz, 1H), 3.72 (t, J=8.8 Hz,1H), 3.56-3.40 (m, 1H), 3.32-3.22 (m, 2H), 2.86-2.61 (m, 2H), 2.47 (td,J=10.0, 5.0 Hz, 1H), 2.34 (s, 3H), 2.32-2.23 (m, 1H), 2.22-2.01 (m, 2H),1.88 (dd, J=9.5, 4.0 Hz, 2H), 1.74 (dt, J=13.4, 3.8 Hz, 1H), 1.50 (s,9H), 1.42-1.30 (m, 1H), 1.09 (dd, J=10.0, 7.0 Hz, 6H). ¹⁹F NMR (376 MHz,methanol-d4) δ −184.32.

Step 3B.(S)-4-((3S,4S)-3-Fluoro-1-((R)-1-(4-methylbenzyl)-2-oxopyrrolidin-3-yl)piperidin-4-yl)phenyl2-amino-3-methylbutanoate Hydrochloride

To a solution of(S)-4-((3S,4S)-3-fluoro-1-((R)-1-(4-methylbenzyl)-2-oxopyrrolidin-3-yl)piperidin-4-yl)phenyl2-((tert-butoxycarbonyl)amino)-3-methylbutanoate (0.025 g, 0.043 mmol)in DCM (1.5 mL) at −20° C. was added HCl in diethyl ether (2.5 ml, 2.50mmol, 1.0 M). The reaction mixture was slowly warmed to rt over 10 minand then allowed to stir at rt for 19 h. The solvent was then removed invacuo to provide a pale yellow semisolid. The crude product was thenpurified by RP-HPLC on a Sunfire C18 (250×20 mm) 5 μm column using agradient of 10% solvent B to 75% solvent B over 12 minutes at 15 mL/minwhere solvent A=0.05% HCl in water and solvent B=acetonitrile. Activefractions were concentrated by lyophilization to provide 10.2 mg (44%)of(S)-4-((3S,4S)-3-fluoro-1-((R)-1-(4-methylbenzyl)-2-oxopyrrolidin-3-yl)piperidin-4-yl)phenyl2-amino-3-methylbutanoate hydrochloride, the titled compound of example2 as an off-white solid. LC-MS (Method A) RT=2.20 min, (M+H)⁺=482.2. ¹HNMR: (400 MHz, DMSO-d6) δ ppm 8.64-8.78 (m, 3H) 7.38-7.46 (m, 2H) 7.23(d, J=8.53 Hz, 2H) 7.18 (s, 4H) 5.04-5.26 (m, 1H) 4.42 (d, J=9.54 Hz,3H) 4.17-4.23 (m, 2H) 3.29-3.40 (m, 4H) 3.19-3.28 (m, 2H) 2.30 (s, 6H)2.04-2.19 (m, 2H) 1.11 (dd, J=12.55, 7.03 Hz, 6H). ¹⁹F NMR (376 MHz,DMSO-d6) δ −183.904.

Example 4.(S)-4-((3S,4S)-3-Fluoro-1-((R)-1-(4-methylbenzyl)-2-oxopyrrolidin-3-yl)piperidin-4-yl)phenyl2-aminopropanoate Hydrochloride

Step 4A.(S)-4-((3S,4S)-3-Fluoro-1-((R)-1-(4-methylbenzyl)-2-oxopyrrolidin-3-yl)piperidin-4-yl)phenyl2-((tert-butoxycarbonyl)amino)propanoate

To a solution of(R)-3-((3S,4S)-3-fluoro-4-(4-hydroxyphenyl)piperidin-1-yl)-1-(4-methylbenzyl)pyrrolidin-2-one(0.03 g, 0.078 mmol, example 1) in DCM (5 mL) was added(S)-2-((tert-butoxycarbonyl)amino)propanoic acid (0.077 g, 0.408 mmol)followed by DCC (0.049 g, 0.235 mmol) and DMAP (9.58 mg, 0.078 mmol).The reaction mixture was stirred at rt for 18 h. Water (15 mL) was thenadded, and the layers were separated. The aqueous layer was extractedwith DCM (3×15 mL) and the organic layers were combined, dried overNa₂SO₄, and concentrated to a crude product. The crude product waspurified by preparative TLC eluting with 20% ethyl acetate in petroleumether to provide the purified product(S)-4-((3S,4S)-3-fluoro-1-((R)-1-(4-methylbenzyl)-2-oxopyrrolidin-3-yl)piperidin-4-yl)phenyl2-((tert-butoxycarbonyl)amino)propanoate (0.032 g, 0.058 mmol, 74%yield) as off-white semi solid. LC-MS (Method A) RT=2.40 min,(M+H)⁺=554.2. ¹H NMR (400 MHz, DMSO-d₆) δ 7.51 (d, J=7.0 Hz, 1H),7.44-7.32 (m, J=8.5 Hz, 2H), 7.21-7.09 (m, 4H), 7.07-6.98 (m, J=8.5 Hz,2H), 4.62 (d, J=4.5 Hz, 1H), 4.39 (d, J=15.1 Hz, 1H), 4.30 (d, J=15.1Hz, 1H), 4.27-4.17 (m, 1H), 3.58 (t, J=8.5 Hz, 1H), 3.50-3.40 (m, 1H),3.22-3.07 (m, 2H), 2.81-2.64 (m, 3H), 2.39-2.31 (m, 1H), 2.29 (s, 3H),2.17-2.04 (m, 1H), 1.93 (dd, J=12.8, 8.3 Hz, 1H), 1.78 (br. s., 1H),1.74-1.59 (m, 1H), 1.41 (s, 9H), 1.39 (d, J=2.5 Hz, 3H). ¹⁹F NMR (376MHz, DMSO-d6) δ −180.172.

Step 4B.(S)-4-((3S,4S)-3-Fluoro-1-((R)-1-(4-methylbenzyl)-2-oxopyrrolidin-3-yl)piperidin-4-yl)phenyl2-aminopropanoate Hydrochloride

To a solution of(S)-4-((3S,4S)-3-fluoro-1-((R)-1-(4-methylbenzyl)-2-oxopyrrolidin-3-yl)piperidin-4-yl)phenyl2-((tert-butoxycarbonyl)amino)propanoate (0.032 g, 0.058 mmol) in DCM (2mL) at −20° C. was added HCl in diethyl ether (2.0 ml, 2.0 mmol, 1.0 M).The reaction mixture was slowly warmed to rt over 10 min and thenallowed to stir at rt for 19 h. The solvent was then removed in vacuo toprovide a pale yellow semisolid. The crude product was then purified byRP-HPLC on a Kinetex C18 (250×20 mm) 5 μm column using a gradient of 10%solvent B to 40% solvent B over 7 minutes at 15 mL/min where solventA=0.05% HCl in water and solvent B=acetonitrile. Active fractions wereconcentrated by lyophilization to provide 4.7 mg (16%) of(S)-4-((3S,4S)-3-fluoro-1-((R)-1-(4-methylbenzyl)-2-oxopyrrolidin-3-yl)piperidin-4-yl)phenyl2-aminopropanoate hydrochloride, the titled compound of example 4 as anoff-white solid. LC-MS (Method A) RT=1.762 min, (M+H)⁺=454. ¹H NMR (400MHz, DMSO-d₆) δ 7.41 (d, J=9.0 Hz, 2H), 7.24-7.10 (m, 6H), 5.10-4.85 (m,1H), 4.45-4.22 (m, 4H), 4.04-3.94 (m, 1H), 3.34-3.18 (m, 4H), 3.06 (d,J=12.0 Hz, 2H), 2.43-2.31 (m, 1H), 2.27 (s, 3H), 2.24-2.14 (m, 1H),2.13-2.01 (m, 1H), 2.01-1.85 (m, 1H), 1.58 (d, J=7.0 Hz, 3H). ¹⁹F NMR(376 MHz, DMSO-d6) δ −183.778.

Example 5.(S)-2-Amino-4-(4-((3S,4S)-3-fluoro-1-((R)-1-(4-methylbenzyl)-2-oxopyrrolidin-3-yl)piperidin-4-yl)phenoxy)-4-oxobutanoicAcid Hydrochloride

Step 5A. (S)-1-tert-Butyl4-(4-((3S,4S)-3-fluoro-1-((R)-1-(4-methylbenzyl)-2-oxopyrrolidin-3-yl)piperidin-4-yl)phenyl)2-((tert-butoxycarbonyl)amino)succinate

To a solution of(R)-3-((3S,4S)-3-fluoro-4-(4-hydroxyphenyl)piperidin-1-yl)-1-(4-methylbenzyl)pyrrolidin-2-one(0.03 g, 0.078 mmol) in DCM (5 mL) was added(S)-4-(tert-butoxy)-3-((tert-butoxycarbonyl)amino)-4-oxobutanoic acid(0.118 g, 0.408 mmol) followed by DCC (0.049 g, 0.235 mmol) and DMAP(9.58 mg, 0.078 mmol). The reaction was stirred at rt for 18 hours.Water (15 mL) was then added, and the layers were separated. The aqueouslayer was extracted with DCM (3×15 mL) and the organic layers werecombined, dried over Na₂SO₄, and concentrated to a crude product. Thecrude product was purified by preparative TLC eluting with 25% ethylacetate in petroleum ether to provide the purified product (37 mg, 68%)as an off-white semi solid. LC-MS (Method A) RT=2.55 min, (M+H)⁺=654.4.¹H NMR (400 MHz, DMSO-d₆) δ 7.46-7.34 (m, 2H), 7.20-6.98 (m, 6H),4.82-4.53 (m, 1H), 4.43-4.24 (m, 2H), 4.11 (d, J=14.1 Hz, 1H), 3.58 (t,J=8.8 Hz, 1H), 3.48-3.39 (m, 1H), 3.22-3.07 (m, 3H), 3.02 (dd, J=16.1,6.5 Hz, 1H), 2.87 (dd, J=15.8, 7.8 Hz, 1H), 2.78-2.63 (m, 2H), 2.38-2.31(m, 1H), 2.29 (s, 3H), 2.17-2.03 (m, 1H), 1.98-1.86 (m, 1H), 1.78 (br.s., 1H), 1.74-1.58 (m, 1H), 1.42 (s, 9H), 1.39 (s, 9H). ¹⁹F NMR (376MHz, DMSO-d6) δ −180.707.

Step 5B.(S)-2-amino-4-(4-((3S,4S)-3-fluoro-1-((R)-1-(4-methylbenzyl)-2-oxopyrrolidin-3-yl)piperidin-4-yl)phenoxy)-4-oxobutanoicAcid Hydrochloride

To a solution of (S)-1-tert-butyl4-(4-((3S,4S)-3-fluoro-1-((R)-1-(4-methylbenzyl)-2-oxopyrrolidin-3-yl)piperidin-4-yl)phenyl)2-((tert-butoxycarbonyl)amino)succinate (0.032 g, 0.049 mmol) in DCM (2mL) at −20° C. was added HCl in diethyl ether (2.0 ml, 2.0 mmol, 1.0 M).The reaction mixture was slowly warmed to rt over 10 min and thenallowed to stir at rt for 19 h. The solvent was then removed in vacuo toprovide a pale yellow semisolid. The crude product was then purified byRP-HPLC on a YMC Triart C18 (150×19 mm) 5 μm column using a gradient of10% solvent B to 40% solvent B over 7 minutes at 15 mL/min where solventA=0.05% HCl in water and solvent B=acetonitrile. Active fractions wereconcentrated by lyophilization to provide 17 mg (57%) of(S)-2-amino-4-(4-((3S,4S)-3-fluoro-1-((R)-1-(4-methylbenzyl)-2-oxopyrrolidin-3-yl)piperidin-4-yl)phenoxy)-4-oxobutanoicacid hydrochloride, the titled compound of example 5 as an off-whitesolid. LC-MS (Method A) RT=1.808 min, (M+H)⁺=498.2 ¹H NMR (400 MHz,DMSO-d₆) δ 7.36 (d, J=8.5 Hz, 2H), 7.21-7.03 (m, 8H), 6.75 (d, J=8.5 Hz,1H), 4.95-4.72 (m, 1H), 4.42-4.24 (m, 3H), 4.10 (t, J=5.3 Hz, 1H),4.07-4.01 (m, 1H), 3.93-3.84 (m, 1H), 3.83-3.68 (m, 1H), 3.33-3.10 (m,5H), 3.04 (br. s., 1H), 2.93 (br. s., 1H), 2.89-2.71 (m, 2H), 2.26 (s,3H), 1.96 (br. s., 1H), 1.82 (br. s., 1H). ¹⁹F NMR (376 MHz, DMSO-d6) δ−180.707.

Example 6.(R)-1-(4-(Difluoromethyl)benzyl)-3-((3S,4S)-3-fluoro-4-(4-hydroxyphenyl)piperidin-1-yl)pyrrolidin-2-one

A solution of (S)-1-(4-(difluoromethyl)benzyl)-2-oxopyrrolidin-3-ylmethanesulfonate (500 mg, 1.57 mmol, intermediate M) in 5.0 mL ofacetonitrile was added dropwise over 1.5 h to a stirred mixture of4-((3S,4S)-3-fluoropiperidin-4-yl)phenol, hydrochloride (363 mg, 1.57mmol, intermediate F) and N,N-diisopropylethylamine (1.09 mL, 6.26 mmol)in 5.0 mL of acetonitrile maintained at 85° C. After complete addition,the reaction mixture was stirred at 85° C. for 16 h. The reactionmixture was concentrated in vacuo. The residue was purified using silicagel column chromatography (0-100% EtOAc/hexanes) to afford adiasteromeric mixture (partial epimerization of the lactam stereocenter)of1-(4-(difluoromethyl)benzyl)-3-((3S,4S)-3-fluoro-4-(4-hydroxyphenyl)piperidin-1-yl)pyrrolidin-2-one(235 mg, 35% yield). A sample of the diastereomeric mixture (780 mg) wasseparated by preparative chiral SFC (column=Lux Cellulose-2 (21×250 mm,5 μm); isocratic solvent=20% methanol (with 15 mM ammonia)/80% CO₂;temp=35° C.; flow rate=60 mL/min; injection volumn=1.0 mL (˜20 mg/mL inMeOH) stacked @ 13 min intervals; λ=210 nM; Peak 1=19.6 min, Peak 2=24.5min) to afford the titled compounds of example 6 (Peak-1, 389 mg) and(S)-1-(4-(difluoromethyl)benzyl)-3-((3S,4S)-3-fluoro-4-(4-hydroxyphenyl)piperidin-1-yl)pyrrolidin-2-one(Peak 2, 242 mg). Data for Example 6: LC-MS m/z 419.3 (M+H⁺); ¹H NMR(500 MHz, chloroform-d) δ 7.50 (d, J=7.9 Hz, 2H), 7.34 (d, J=7.9 Hz,2H), 7.15 (d, J=8.5 Hz, 2H), 6.91-6.80 (m, 2H), 6.65 (t, J=56.4 Hz, 1H),4.96 (s, 1H), 4.77-4.43 (m, 3H), 3.68 (t, J=8.8 Hz, 1H), 3.42-3.33 (m,1H), 3.29-3.14 (m, 2H), 2.85 (d, J=10.4 Hz, 1H), 2.78-2.69 (m, 1H),2.69-2.57 (m, 1H), 2.48 (td, J=9.9, 4.9 Hz, 1H), 2.21-2.11 (m, 1H), 2.04(dq, J=13.0, 8.6 Hz, 1H), 1.94-1.82 (m, 2H)). The relative and absoluteconfiguration of Example 114, P-1 was confirmed by single crystal X-rayanalysis.

Example 7.4-((3S,4S)-3-fluoro-1-((R)-1-(4-methylbenzyl)-2-oxopyrrolidin-3-vl)piperidin-4-yl)phenylDihydrogen Phosphate

To a suspension of(R)-1-(4-(difluoromethyl)benzyl)-3-((3S,4S)-3-fluoro-4-(4-hydroxyphenyl)piperidin-1-yl)pyrrolidin-2-one(100 mg, 0.239 mmol, from example 6) in dichloromethane (10 mL) wasadded triethylamine (0.233 ml, 1.67 mmol) at −20° C. To the chilledsolution was added POCl₃ (0.111 ml, 1.20 mmol) at −20° C., and then thereaction mixture was stirred for 2-3 hours at −20° C. Water (10 mL) wasadded and the mixture was stirred for 1.5 h. The mixture was extractedwith dichloromethane. The organic layers was dried over sodium sulfate,filtered, and concentrated in vacuo. The crude product was purified byreverse phase preparatory HPLC on a LUNA C8 (250 mm×19 mm ID) 5 μmcolumn eluting with a gradient of solvent A=10 mM ammonium acetate inwater pH 4.5 and solvent B=acetonitrile. The titled compound of example7 (21 mg, 18%) was isolated from the appropriate fractions bylyophilization as a white solid. LCMS (M+H)⁺=499.2; ¹H NMR (400 MHz,METHANOL-d4) δ ppm 7.55 (d, J=8.03 Hz, 2H) 7.41 (d, J=8.03 Hz, 2H) 7.21(s, 4H) 6.62-6.92 (m, 1H) 4.51-4.64 (m, 3H) 3.76 (t, J=8.78 Hz, 1H)3.43-3.51 (m, 1H) 3.36 (d, J=6.02 Hz, 1H) 3.26-3.30 (m, 1H) 2.81 (br.s., 1H) 2.70-2.78 (m, 1H) 2.59-2.69 (m, 1H) 2.48 (td, J=9.91, 4.77 Hz,1H) 2.17-2.27 (m, 1H) 2.06-2.15 (m, 1H) 1.80-1.89 (m, 2H).

Example 8.(R)-1-(4-Fluorobenzyl)-3-(4-(4-hydroxyphenyl)piperidin-1-yl)pyrrolidin-2-one

To a solution of1-(4-fluorobenzyl)-3-(4-(4-methoxyphenyl)piperidin-1-yl)pyrrolidin-2-one(3 g, 7.9 mmol, intermediate S) in dry dichloromethane (100 mL) under aN₂ atmosphere at −78° C. was added 1 M borontribromide indichloromethane (39 mL, 39 mmol) and the resulting mixture was allowedto warm up to room temperature over 3 h, with stirring. The reaction wasquenched with water (30 mL) and the organic layer was separated, washedwith water and brine, and concentrated. The crude product was purifiedby flash chromatography on silica gel using 15% EtOAc in petroleum etherto yield racemic1-(4-fluorobenzyl)-3-(4-(4-hydroxyphenyl)piperidin-1-yl)pyrrolidin-2-one(2.1 g, 73% yield); ¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.49-1.74 (m, 4H)1.90-2.11 (m, 2H) 2.24-2.42 (m, 2H) 2.65-2.80 (m, 2H) 2.99-3.23 (m, 3H)3.40-3.54 (m, 1H) 4.27-4.46 (m, 2H) 6.61-6.70 (m, 2H) 6.95-7.04 (m, 2H)7.17-7.31 (m, 4H) 9.10-9.16 (m, 1H). LCMS (ES-API) 369.2 m/z (M+H)⁺. Aportion of the racemate (40 mg) was separated via SFC on a Chiralpak-IA250 mm×4.6 mm, 5 μm column eluting with 35% solvent B, where solventA=CO₂ and solvent B=0.3% DEA in methanol at a total flow of 3 mL/min.Peak 1 showed a RT of 4.35 min (11 mg) and Peak 2 showed a RT of 6.29min (13 mg). Data for example 8 (Peak 2): LC/MS (M+H)⁺=369.2; ¹H NMR(400 MHz, DMSO-d₆) δ ppm 1.47-1.59 (m, 1H) 1.65-1.75 (m, 1H) 1.84-1.96(m, 1H) 2.03-2.12 (m, 1H) 2.24-2.43 (m, 1H) 2.63-2.72 (m, 2H) 2.72-2.85(m, 2H) 2.96-3.05 (m, 2H) 3.09-3.23 (m, 2H) 3.41-3.54 (m, 1H) 4.23-4.50(m, 2H) 6.58-6.71 (m, 2H) 6.96-7.10 (m, 2H) 7.15-7.21 (m, 2H) 7.26-7.34(m, 2H) 9.06-9.19 (m, 1H).

Example 9.(R)-4-(1-(1-(4-fluorobenzyl)-2-oxopyrrolidin-3-yl)piperidin-4-yl)phenylDihydrogen Phosphate

Phosphorus oxychloride (1.27 mL, 13.6 mmol) was added to a round bottomflask charged with THF (10 mL). The solution was cooled below 0° C.using an ice/methanol bath. A suspension of(R)-1-(4-fluorobenzyl)-3-(4-(4-hydroxyphenyl)piperidin-1-yl)pyrrolidin-2-one(1.00 g, 2.71 mmol, example 8) in THF (18 mL) was added. After 5 min,triethylamine (0.946 mL, 6.79 mmol) was added slowly at a bathtemperature below 5° C. The reaction mixture was stirred at 0° C. for 90min. A solution of 1 N aqueous sodium hydroxide (8.69 mL, 8.69 mmol) wasadded dropwise. The pH was measured to be ˜0. The mixture was allowed towarm to rt and stir for 3 h. The crude reaction mixture was concentratedin vacuo at <30° C. to afford a clear solution. The solution wastriturated with 1 N aqueous NaOH to pH ˜1. The mixture was cooled in anice bath. A semi-solid crashed out. All liquid was decanted off. Thesemi-solid was suspended in 90% ethanol and then a collected by vacuumfiltration. The product was presumed to be the HCl salt of(R)-4-(1-(1-(4-fluorobenzyl)-2-oxopyrrolidin-3-yl)piperidin-4-yl)phenyldihydrogen phosphate (560 mg, 42%). A solution of 25% sodium methoxidein methanol (250 mg, 1.16 mmol) was added to a slurry of(R)-4-(1-(1-(4-fluorobenzyl)-2-oxopyrrolidin-3-yl)piperidin-4-yl)phenyldihydrogen phosphate, HCl (560 mg, 1.16 mmol) in methanol. The mixturewas stirred until clear and then concentrated in vacuo. The residue wasdissolved in 90% ethanol/water and chilled in the freezer. The solidprecipitate was collected using vacuum filtration. The solid was driedunder high vacuum to afford the titled compound of example 9 (230 mg,19% yield): LC/MS (M+H)⁺=449.2; ¹H NMR (500 MHz, methanol-d₄) δ7.35-7.25 (m, 2H), 7.21-7.11 (m, 4H), 7.11-7.03 (m, 2H), 4.55-4.35 (m,2H), 3.61 (t, J=8.8 Hz, 1H), 3.30-3.20 (m, 2H), 3.19-3.11 (m, 1H),2.92-2.84 (m, 1H), 2.75 (td, J=11.1, 3.5 Hz, 1H), 2.55-2.41 (m, 2H),2.23-2.13 (m, 1H), 2.12-2.00 (m, 1H), 1.84-1.70 (m, 4H); ³¹P NMR (202MHz, methanol-d₄) δ ppm −3.38.

Biological Methods

Radioligand Binding Assay.

Binding experiments to determine binding to NR2B-subtype NMDA receptorswere performed on forebrains of 8-10 weeks old male Sprague Dawley rats(Harlan, Netherlands) using ³H Ro 25-6981 (Mutel V; Buchy D;Klingelschmidt A; Messer J; Bleuel Z; Kemp J A; Richards J G. Journal ofNeurochemistry, 1998, 70(5):2147-2155. Rats were decapitated withoutanesthesia using a Guillotine (approved by animal ethics committee) andthe harvested brains were snap-frozen and stored at −80° C. for 3-6months for membrane preparation.

For membrane preparation, rat forebrains were thawed on ice for 20minutes in homogenization buffer composed of 50 mM KH₂PO₄ (pH adjustedto 7.4 with KOH), 1 mM EDTA, 0.005% Triton X 100 and protease inhibitorcocktail (Sigma Aldrich). Thawed brains were homogenized using a Douncehomogenizer and centrifuged at 48000×g for 20 min. The pellet wasresuspended in cold buffer and homogenized again using a Douncehomogenizer. Subsequently, the homogenate was aliquoted, snap-frozen andstored at −80° C. for not more than 3-4 months.

To perform the competition binding assay, thawed membrane homogenate wasadded to each well of a 96-well plate (20 μg/well). The experimentalcompounds were serially diluted in 100% DMSO and added to each row ofthe assay plate to achieve desired compound concentrations, keeping theDMSO concentration in the assay plate at 1.33% of the final reactionvolume. Next, ³H Ro 25-6981 (4 nM) was added to the assay plate. Afterincubation for 1 hr at room temperature, the membrane bound radioligandwas harvested on to GF/B filter plates (treated with 0.5% PEI for 1 hrat room temperature). The filter plates were dried at 50° C. for 20mins, incubated with microscint 20 for 10 minutes and finally, thecounts were read on TopCount (Perkin Elmer). Non-specific binding wasdetermined using MK-0657 (the preparation of this compound is describedas example 1 in WO 2004 108705 (40 μM). CPM values were converted to %inhibition and the concentration response curves were plotted usingcustom made software. Each experiment was repeated at least twice toobtain the final binding K_(i) values for experimental compounds. Usingthis assay, the compound of example 1 showed a binding Ki of 4 nM, thecompound of example 6 showed a binding Ki of 4 nM, the compound ofexample 8 showed a binding Ki of 1.4 nM.

Ex Vivo Occupancy Assay.

This assay demonstrates that the compound of example 1 occupiesbrain-resident NR2B-subtype receptors in animals after dosing. 7-9 weeksold male CD-1 mice were dosed intravenously in a vehicle consisting of10% dimethylacetamide, 40% PEG-400, 30% hydroxypropyl betacyclodextrin,and 30% water with experimental compounds and the forebrains wereharvested 15 minutes post-dosing by decapitation. The brain samples wereimmediately snap-frozen and stored at −80° C. On the following day, thedosed brain samples were thawed on ice for 15-20 minutes followed byhomogenization using Polytron for 10 seconds in cold homogenizationbuffer composed of 50 mM KH₂PO₄ (pH adjusted to 7.4 with KOH), 1 mMEDTA, 0.005% Triton X 100 and protease inhibitor cocktail (SigmaAldrich). The crude homogenates were further homogenized using a Douncehomogenizer and the homogenized membrane aliquots from all animals wereflash-frozen and stored at −80° C. until further use. The wholehomogenization process was performed on ice.

For determining occupancy, the membrane homogenates were first thawed onice and then needle-homogenized using a 25 gauge needle. The homogenizedmembrane (6.4 mg/ml) was added to a 96-well plate followed by additionof ³H Ro 25-6981 (6 nM). The reaction mixture was incubated for 5minutes on a shaker at 4° C. and then harvested onto GF/B filter plates(treated with 0.5% PEI for 1 hr at room temperature). The filter plateswere dried at 50° C. for 20 mins, incubated with microscint 20 for 10minutes and read on TopCount (Perkin Elmer). Each dose or compound groupconsisted of 4-5 animals. The control group of animals was dosed withvehicle alone. Membrane from each animal was added in triplicates to theassay plate. Non-specific binding was determined using 10 μM Ro 25-6981added to the wells containing membrane homogenates from vehicle-dosedanimals. Specific counts/minute was converted to % occupancy at eachdose of a compound for each animal using the following equation:

${\% \mspace{14mu} {Occupancy}\mspace{14mu} ( {{animal}\mspace{14mu} A} )} = {100 - ( {\frac{{specific}\mspace{20mu} {CPM}\mspace{14mu} {of}\mspace{14mu} {animal}\mspace{14mu} A}{{Average}\mspace{14mu} {CPM}\mspace{14mu} {from}\mspace{14mu} {control}\mspace{14mu} {group}} \times 100} )}$

Using this procedure, the compound of example 1 showed 95% NR2B receptoroccupancy after a 3 mg/Kg i.v. dose. Drug levels were determined by massspectroscopy in the usual manner. Drug levels in the blood plasma were1106 nM in at this dose, and drug levels in the homogonized brain tissuewere 1984 nM. The compound of example 6 showed 97% NR2B receptoroccupancy after a 3 mg/Kg i.v. dose. Drug levels in the blood plasmawere 1800 nM in at this dose, and drug levels in the homogonized braintissue were 2200 nM. The compound of example 8 showed 96% NR2B receptoroccupancy after a 3 mg/Kg i.v. dose. Drug levels in the blood plasmawere 570 nM at this dose, and drug levels in the homogonized braintissue were 900 nM.

hERG Electrophysiology Assay.

The experimental compounds were assessed for hERG activity on HEK 293cells stably expressing hERG channels using patch clamp technique.Coverslips plated with hERG expressing cells were placed in theexperimental chamber and were perfused with a solution composed of (inmM): 140 NaCl, 4 KCl, 1.8 CaCl₂), 1 MgCl₂, 10 Glucose, 10 HEPES (pH 7.4,NaOH) at room temperature. Borosilicate patch pipettes had tipresistances of 2-4 Mohms when filled with an internal solutioncontaining: 130 KCl, 1 MgCl₂, 1 CaCl₂, 10 EGTA, 10 HEPES, 5 ATP-K₂ (pH7.2, KOH). The cells were clamped at −80 mV in whole cell configurationusing an Axopatch 200B (Axon instruments, Union City, Calif.) patchclamp amplifier controlled by pClamp (Axon instruments) software. Uponformation of a gigaseal, the following voltage protocol was repeatedly(0.05 Hz) applied to record tail currents: depolarization step from −80mV to +20 mV for 2 seconds followed by a hyperpolarization step to −65mV (3 seconds) to elicit tail currents and then, back to the holdingpotential. Compounds were applied after stabilization of tail current.First, tail currents were recorded in presence of extracellular solutionalone (control) and subsequently, in extracellular solution containingincreasing compound concentrations. Each compound concentration wasapplied for 2-5 minutes. The percentage inhibition at each concentrationwas calculated as reduction in peak tail current with respect to thepeak tail current recorded in the presence of control solution. Dataanalysis was performed in custom made software. The percent inhibitionsat different concentrations were plotted to obtain a concentrationresponse curve, which was subsequently fitted with a four parameterequation to calculate the hERG IC₅₀ value. Using this procedure, thecompound of example 1 is a poor inhibitor of the hERG channel, with anIC₅₀=28 μM. The compound of example 6 is a poor inhibitor of the hERGchannel, with an IC₅₀=13.5 μM.

Mouse Forced Swim Test (mFST).

Forced Swim Test (FST) is an animal model used to assess antidepressantcompounds in preclinical studies. The FST was performed similar to themethod of Porsolt et al. with modifications (Porsolt R D, Bertin A,Jalfre M. Behavioral despair in mice: a primary screening test forantidepressants. Arch Int Pharmacodyn Thér 1977; 229:327-36). In thisparadigm, mice are forced to swim in an inescapable cylinder filled withwater. Under these conditions, mice will initially try to escape andeventually develop immobility behavior; this behavior is interpreted asa passive stress-coping strategy or depression-like behavior. Swim tankswere positioned inside a box made of plastic. Each tank was separatedfrom each other by opaque plastic sheets to the height of cylinders.Three mice were subjected to test at a time. Swim sessions wereconducted for 6 min by placing mice in individual glass cylinders (46 cmheight×20 cm diameter) containing water (20-cm deep, maintained at24-25° C.). At this water level, the mouse tail does not touch thebottom of the container. The mouse was judged to be immobile whenever itremained floating passively without struggling in the water and onlymaking those movements necessary to keep its nose/head above the waterand to keep it afloat. The duration of immobility was evaluated duringthe total 6 min of the test and expressed as duration (sec) ofimmobility. Each mouse was tested only once. At the end of each session,mice were dried with a dry cloth and returned to their home cage placedon a thermal blanket to prevent hypothermia. Water was replaced aftereach trial. All testing sessions were recorded with a video camera (SonyHandicam, Model: DCR-HC38E; PAL) and scoring was done using the ForcedSwim Scan, Version 2.0 software (Clever Systems Inc., Reston, Va., USA;see Hayashi E, Shimamura M, Kuratani K, Kinoshita M, Hara H. Automatedexperimental system capturing three behavioral components during murineforced swim test. Life Sci. 2011 Feb. 28; 88(9-10):411-7 and Yuan P,Tragon T, Xia M, Leclair C A, Skoumbourdis A P, Zheng W, Thomas C J,Huang R, Austin C P, Chen G, Guitart X. Phosphodiesterase 4 inhibitorsenhance sexual pleasure-seeking activity in rodents. Pharmacol BiochemBehav. 2011; 98(3):349-55). For NCE testing: Test compound wasadministered in mice 15 min before swim session by i.v. route andimmobility time was recorded for next 6 min. At the end of FST, themouse were euthanized by rapid decapitation method and plasma and brainsamples were collected and stored under −80° C. till further analysis.In the mouse forced swim assay, the compound of example 1 was dosedintravenously in a vehicle of 30% hydroxypropyl betacyclodextrin/70%citrate buffer pH 4 at a 5 mL/Kg dosing volume. The compound of example1 demonstrated a statistically significant decrease in immobility timeat 1 mg/Kg under these conditions. Drug levels were 268+/−128 nM in theplasma and 749+/−215 nM in the brain at this dose. The NR2B receptoroccupancy was determined as reported above and was determined to be 73%.The compound of example 6 demonstrated a statistically significantdecrease in immobility time at 1 mg/Kg under these same conditions. Druglevels were 360 nM in the plasma. The NR2B receptor occupancy wasdetermined to be 79%.

We claim:
 1. A pharmaceutical composition comprising the compound4-((3S,4S)-3-fluoro-1-((R)-1-(4-methylbenzyl)-2-oxopyrrolidin-3-yl)piperidin-4-yl)phenyldihydrogen phosphate or a pharmaceutically acceptable salt thereof and apharmaceutically acceptable carrier.